The Role of Reticulocyte Hemoglobin Content in Diagnosing Iron Deficiency in Childhood Cancer

Abstract Background  The prevalence of iron deficiency (ID) and iron deficiency anemia (IDA) in children with cancer is not well studied. The detection of ID and IDA using sensitive laboratory tools may facilitate early diagnosis and treatment in this cohort. In this regard, reticulocyte hemoglobin (Ret-He) content serves as a cost-effective measurement that remains unaffected by inflammation, unlike the ferritin test. Aim  The objective of this study is to analyze the role of Ret-He as a diagnostic tool to identify functional and absolute ID and IDA in children with cancer. Methods  We conducted a cross-sectional study in children aged 0 to 18 years. Blood samples were collected to compare Ret-He values with iron status, reflected by hemoglobin (Hb), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), serum iron (SI), total iron binding capacity (TIBC), and ferritin and transferrin saturation. The overall discriminative power of Ret-He in detecting ID and IDA was assessed using receiver operating characteristic analysis. Results  Of the 135 children included in the study, 58 (43.0%) had anemia. Among them, 20 (14.8%) had IDA (8 [5.9%] absolute and 12 [8.9%] functional), while 25 (18.5%) had ID (16 [11.9%] absolute and 9 [6.7%] functional). The Ret-He value was significantly related to iron status ( p ≤ 0.002). Ret-He was also shown to have a significant correlation with the abovementioned hematological parameters ( p  = 0.000), except TIBC. Multivariate analysis revealed a significant relationship between Hb ( p  = 0.051), MCH ( p  = 0.000), and MCHC ( p  = 0.001) and Ret-He. Ret-He values of 33.7, 32.7, 32.4 and 28.6 pg were established as optimal cut-off values to identify functional ID, absolute ID, functional IDA, and absolute IDA, respectively. Conclusion  Ret-He is a reliable diagnostic tool for absolute and functional IDA in children with cancer.


Introduction
Children suffering from chronic diseases, such as cancer, are more susceptible to both iron deficiency (ID) and iron deficiency anemia (IDA).A study conducted by the European Anemia in cancer patients can arise from factors like malnutrition, malabsorption, chronic inflammation, bleeding, therapy-induced myelosuppression, bone marrow infiltration, hemolysis, hypersplenism, and ID.The disrupted iron homeostasis and metabolism in cancer patients are primarily due to chronic inflammation, which leads to iron sequestration in macrophages, causing limited iron availability for red blood cell production in the bone marrow. 3,4DA can adversely affect physical performance, leading to general weakness and fatigue and potentially reducing the effectiveness of chemotherapy/radiotherapy against tumors. 3Thus, the early detection of ID is crucial to address it with simple treatments like iron supplementation or erythropoietin and limit the need for packed red cell transfusion in cancer patients.
Although the gold-standard diagnostic tool for ID is bone marrow staining with Prussian blue, this method is invasive and expensive. 5In 2010, the American Academy of Pediatrics (AAP) stated that ID can be diagnosed by evaluating ferritin and c-reactive protein levels or measuring reticulocyte hemoglobin (Ret-He), with low hemoglobin levels indicating IDA. 6However, ferritin is an acute-phase protein that can increase under inflammatory conditions, including malignancy.The European Society for Medical Oncology (ESMO) guidelines define ID in cancer patients as ferritin levels <100 ng/mL or transferrin saturation (TS) < 20%. 3,5n recent years, the potential of Ret-He content as an early marker for ID has been highlighted.Reticulocytes are immature erythrocytes released from the bone marrow that can reflect the erythropoiesis status over the preceding 3 to 4 days. 7,8Unlike ferritin, Ret-He is not influenced by inflammation as it is not an acute-phase protein. 6,9The hemoglobin content in reticulocytes can be assessed through measures such as Ret-He content (CHr or Ret-He), both utilizing flow cytometry and reported in picograms. 8,10,11The Ret-He laboratory test can be performed alongside routine blood tests without the need for additional blood samples. 9,123][14] In this study, we investigated the diagnostic value of Ret-He in identifying ID and IDA in children with cancer to facilitate the simple detection of these conditions.

Subjects
A cross-sectional study was conducted in Cipto Mangunkusumo Hospital from March to June 2021.Hospitalized and outpatient children aged 0 to 18 years with cancer were selected as participants.Patients with a history of iron therapy or blood transfusion in the past month were excluded.Written consent and assent were obtained from the subjects' parents or legal guardians and adolescent patients.

Inclusion and Exclusion Criteria
The inclusion criteria for this study comprised children between the ages of 0 and 18 years with cancer who were either hospitalized or received outpatient treatment.Patients who received iron therapy or blood transfusion within the past month were excluded.No oral iron therapy was initiated for IDA patients.

Laboratory Methods
Venous blood samples (6 mL) were obtained from the subjects.Iron parameters, including hemoglobin (Hb), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), reticulocyte hemoglobin (Ret-He), ferritin, serum iron (SI), and total iron binding capacity (TIBC), were measured via standard techniques.TS was calculated using the formula SI/TIBC Â 100.All parameters were analyzed in the Clinical Pathology Laboratory of Cipto Mangunkusumo Hospital.

Iron Status Definition
The World Health Organization defines anemia as a low Hb value according to age: Hb <11 g/dL in children aged 6 to 59 months, <11.5 in 5 to 11-year-olds, <12 g/dL in 12 to 14year-olds, <12 g/dL in unpregnant girls aged !15 years, and <13 g/dL in boys aged !15 years. 15In this study, ESMO criteria were used to evaluate iron status in children: (1) absolute IDA with low Hb and ferritin <100 ng/mL, (2) functional IDA with low Hb and TS <20% and normal ferritin !100 ng/mL, (3) absolute ID with normal Hb and ferritin <100 ng/mL, and (4) functional ID with normal Hb and TS <20% and ferritin !100 ng/mL. 16

Primary and Secondary Outcomes
The primary outcome of this study was the establishment of optimal Ret-He cut-off values for different types of absolute and functional ID or IDA, with their respective sensitivities, specificities, and predictive values.The secondary outcome was the evaluation of iron status in children with cancer, including the prevalence of ID and IDA.Laboratory indices, such as Hb, MCV, MCH, MCHC, SI, ferritin and TS, and their relationship with Ret-He, were also analyzed.

Statistical Analysis
The correlation between iron status and Ret-He was determined with analysis of variance (ANOVA) or the Kruskal-Wallis test, depending on the data distribution.Normality was assessed using the Kolmogorov-Smirnoff test.ANOVA with Tukey's post-hoc analysis was performed.Ret-He was also compared with other laboratory parameters through correlation analysis using the Pearson and nonparametric Spearman methods.Significant variables were subsequently subjected to multivariate analysis using linear regression.The overall discriminative power of Ret-He to detect iron depletion, ID, and IDA was assessed using receiver operating characteristic (ROC) analysis.Cut-off values were determined for each iron status using Youden's index, where (sensitivity þ specificity) -1 had the highest value.A p-value of <0.05 was considered statistically significant.
The Ethics Committee of the Faculty of Medicine, University of Indonesia, Cipto Mangunkusumo Hospital, approved this study (No.KET-1010/UN2.F1/ETIK/PPM.00.02/2020) on September 14, 2020.This study did not involve any animals.All the research methods involving humans were performed according to the ethical guidelines established by the responsible committee overseeing human experimentation at the institutional and national levels.They also complied with the 1975 Helsinki Declaration, updated in 2013.

Results
A total of 146 children were initially included in this study.Eleven subjects had incomplete data and were excluded; thus, the final study population comprised 135 children (►Supp.Fig. 1).The characteristics of these subjects are shown in ►Table 1.

Iron Status in Children with Cancer
In this study, 58 children (43.0%) had anemia.The prevalence of IDA was 14.8% (20/135), while anemia in the remaining subjects had other causes.Absolute IDA was found in 8 subjects and functional IDA in 12 subjects.The prevalence of ID was 18.5% (25/135).Absolute ID was found in 16 subjects and functional ID in 9 subjects.Analysis of laboratory indices showed that Hb, MCH, MCHC, Ret-He, SI, and TS were statistically significantly related to iron status.All the laboratory parameters assessing iron status in the abovementioned subgroups were statistically significant except ferritin, MCV, and TIBC (►Table 2).

Discussion
The prevalence of anemia in our pediatric cancer study was 43.0%, comparable to a study by ECAS (39%). 1 In this study, the overall prevalence of IDA was 14.8%, similar to prior studies in healthy school-aged children in Jakarta (13 12 and 14% 17 ).Notably, no previous study has examined the prevalence of anemia and ID in children with cancer in Indonesia.
In cancer patients, iron metabolism and regulation are altered due to chronic disease, chronic blood loss, nutritional deficiency, increased consumption by cancer cells, myelosuppressive chemotherapy, and metastases.ID can contribute to DNA damage, genomic instability, and immunological dysfunction during cancer development. 18The timely diagnosis and treatment of ID are crucial in cancer patients to prevent complications associated with anemia, such as impaired exercise capacity, fatigue, reduced quality of life, and an overall poor prognosis. 4,7,18While functional ID is typically predominant, 4,16,[18][19][20] absolute ID was more prevalent in this study, indicating reduced iron stores as the main cause.Thus, restoring iron stores through appropriate therapies is essential.
Iron status assessment in cancer patients remains challenging due to the lack of a gold standard and the impact of inflammatory conditions on standard biochemical tests such as SI and ferritin.According to the AAP, Ret-He is the strongest predictor for ID in children. 6It remains stable compared with other markers and is unaffected by conditions like infection, inflammation, and malignancy. 6,7,21n our study, Ret-He showed a significant positive correlation with other hematological parameters (p ¼ 0.000) except for TIBC.Multivariate analysis revealed a significant relationship between Ret-He and Hb (p ¼ 0.051), MCH (p ¼ 0.000), and MCHC (p ¼ 0.001).The simultaneous analysis of all laboratory parameters in multivariate analysis allows for assessing the effects of variables, as each laboratory parameter represents a specific definition of ID.
Unfortunately, there is no universal cut-off value or guidelines for Ret-He in diagnosing ID or IDA.Prior studies have    suggested various cut-offs, ranging from 25 to 29 pg, [22][23][24] with sensitivities between 70 and 94% and specificities from 72 to 80% in healthy children.Population studies in healthy Indonesian children aged 6 to 18 years and 6 to 12 years found cut-offs of 27.8 12 and 27.8 pg, 14 respectively.In cancer patients, one study in adolescents and adults aged 11 to 94 years reported a higher Ret-He cut-off of 32.0 pg for ID. 7 In our study, cut-offs for functional ID, absolute ID, functional IDA, and absolute IDA were 33.7, 32.7, 32.4, and 28.6 pg, respectively.Studies in children on hemodialysis reported cut-off values of 28.9 25 and 29.0 pg. 26Both these values are more similar to the cut-offs in healthy children.Besides determining the optimal fixed values to evaluate iron status, we also analyzed a range of cut-offs for clinical utility.We found that higher cut-offs (33.Ret-He proved to be a reliable diagnostic tool for functional ID, absolute ID, functional IDA, and absolute IDA, with respective AUCs of 72.4,77.8, 69.7, and 73.1%.It exhibited the highest diagnostic performance in the absolute ID group, demonstrating high sensitivity and specificity.In the functional ID and absolute ID groups, sensitivity was higher than specificity, indicating its reliability as a screening tool.In the functional and absolute IDA groups, specificity was higher, making Ret-He a reliable diagnostic tool supported by a good negative predictive value.Ret-He has been reported as superior in diagnosing ID in children by Brugnara et al, 23 Andriastuti et al, 12 and Syed et al. 27 Using Ret-He as a diagnostic tool can reduce the need for additional iron studies, improving cost-effectiveness and patient comfort. 28,29ur study has limitations that could potentially introduce bias, such as the restriction of the population to subjects who had not received blood transfusions within the past month and the lack of assessment of transfusion frequency and volume.However, from a clinical perspective, the test can effectively be used as a diagnostic tool either at baseline or for new cases, considering the high prevalence of IDA.This study is the first to report the prevalence of ID and IDA in Indonesian children with cancer.Additionally, it is the first to compare Ret-He to other laboratory parameters as a diagnostic tool for pediatric cancer in Indonesia.Further cohort studies are needed to evaluate Ret-He after iron therapy and explore its impact on anemic and irondeficient children with cancer, including newly diagnosed patients.

Conclusion
The prevalence of IDA and ID in childhood cancer in this study was 14.8 and 18.5%, respectively.Ret-He emerged as a reliable diagnostic tool, showing a significant positive correlation with other hematological parameters.Given the burden of IDA in children, it is important to understand its impact on children diagnosed with cancer.The relationship between IDA and cancer in this context is currently understudied and requires further exploration.The present study provides valuable insights into iron metabolism in cancer.It also supports the existing evidence that Ret-He remains unaffected by inflammation in cancer.However, further research is needed to determine the clinical utility of these tests in this population.

Patient Consent
Patient consent was obtained from every subject.

Table 2
Comparison of iron status Note: Data are presented as mean AE standard deviation or median (min-max).a Kruskal-Wallis for nonparametric analysis as alternative to ANOVA test.

Table 3
Correlation analysis between Ret-He and hematological parameters Abbreviations: Hb, hemoglobin; MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration; MCV, mean corpuscular volume; Ret-He, reticulocyte hemoglobin; SI, serum iron; TIBC, total iron binding capacity.Note: Remaining data are analyzed with nonparametric Spearman.a Data are analyzed using Pearson correlation.

Table 4
Multivariate logistic regression analysis between Ret-He and hematological parameters

Table 5
Ret-He cut-off to evaluate iron status Abbreviations: AUC, area under the curve; ID, iron deficiency; IDA, iron deficiency anemia, a Optimal cut-off based on the highest Youden index.